Genetic Toxicology Studies - Study Design of Mammalian Erythrocyte Micronucleus Test (OECD 474)
The Mammalian Erythrocyte Micronucleus Test (OECD 474) is a widely used in vivo genotoxicity assay employed to assess the potential of a substance to induce damage to the chromosomes or the mitotic apparatus of erythroblasts (red blood cell precursors) in bone marrow. It is standardized under the OECD Guideline 474 and plays a crucial role in ensuring the safety of chemicals, pharmaceuticals, food additives, and other agents before they reach the market.
Objectives:
- Determine if a test substance can induce micronuclei (acentric chromosome fragments or whole chromosomes) in the cytoplasm of mature red blood cells (reticulocytes) of animals.
- Evaluate the dose-response relationship for micronucleus induction.
- Classify the substance according to its clastogenic and/or aneugenic potential for regulatory purposes.
Test System:
- Animals: Rodents (usually mice or rats) are used as test subjects.
- Administration: The test substance is administered to the animals by various routes (e.g., oral gavage, intraperitoneal injection) at different doses.
- Exposure duration: Animals are exposed to the substance for a defined period, typically ranging from a single dose to multiple doses over several days.
- Bone marrow and blood sampling: Bone marrow and/or peripheral blood samples are collected at different time points after exposure, typically 24-48 hours.
- Slide preparation and scoring: Extracted cells are stained and visualized under a microscope. A defined number of erythrocytes (typically 1000 per animal) are scored for the presence of micronuclei.
Micronucleus Scoring:
- Reticulocytes (immature red blood cells) are stained and visualized under a microscope.
- A defined number of cells (typically 1000 per dose level) are scored for the presence of micronuclei.
- Micronucleus frequency is calculated as the percentage of cells containing micronuclei
Endpoints
- Micronucleus frequency at different dose levels.
- Dose-response relationship for micronucleus induction.
- Relative reticulocyte count as a measure of bone marrow toxicity.
- Nucleoplasmic ratio (NR) to assess the potential for aneugenic effects.
- Classification of the substance according to OECD 474 criteria (e.g., clastogenic, aneugenic, non-genotoxic).
Benefits:
- Relatively simple and cost-effective compared to other in vivo tests.
- Sensitive in detecting chromosomal damage in mature red blood cells.
- Can be adapted to assess a wide range of chemicals.
- Used in a tiered testing strategy for genotoxicity assessment.
Limitations:
- Does not directly assess damage in germ cells.
- May not capture all types of genetic damage (e.g., point mutations).
- Requires animal sacrifice and ethical considerations.
Additional Resources
- OECD Test Guideline 474:
https://www.oecd.org/chemicalsafety/test-no-474-mammalian-erythrocyte-micronucleus-test-9789264264762-en.htm - Test No. 474: Mammalian Erythrocyte Micronucleus Test | en | OECD:
https://www.oecd.org/env/test-no-474-mammalian-erythrocyte-micronucleus-test-9789264264762-en.htm - The Micronucleus Test for Genotoxicity Detection: A Review :
ttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598904/
